Autologous hematopoietic stem cell transplantation (HSCT) has been an important treatment option for many patients with hematologic malignancies and solid tumors such as acute myeloid leukemia, myelodysplastic syndromes, chronic myeloid leukemia, systemic mastocytosis and breast cancer. During the treatment, anti CD34 antibodies are used to enrich the CD34+ hematopoietic stem cells from mobilized peripheral blood, which are re-infused after a high-dose radio/chemotherapy. Unfortunately, due to the large sample volumes required to obtain sufficient number of stem cells (100-300 mL), the antibody reagents are prohibitively expensive, typically in excess of $10,000 per therapy. To address this problem, we propose to develop an alternate, nucleic acid-based affinity reagents (i.e. aptamers) against the CD34 antigen, which will be stable, exhibit high affinities and specificities, significantly less immunogenic, and considerably less expensive (~10X less) compared to protein-based reagents. Toward this end, we propose three specific aims: first, in order to circumvent the traditional time-consuming and labor-intensive SELEX process, we will adopt and optimize our high throughput microfluidic aptamer discovery platform (Pro-SELEX) to isolate high affinity aptamers (Kd <1 nM) for the CD34 antigen. The selection will be carried out using purified extracellular domain of the CD34 protein from mammalian cell expression system (Chinese Hamster Ovary cells) such that the structure and post-translational modifications are conserved. Secondly, we note that the overwhelming reason for treatment failure is highly correlated to the degree of contamination of circulating tumor cells during the purification of CD34+ cells. Thus, in order to significantly raise the specificity of the aptamers against tumor cells, we will develop a novel, "cell-surface counter selection process" using live leukemia and other tumor cells. Finally, we will evaluate and compare the performance of the anti-CD34 aptamers with commercially available anti-CD34 antibodies. We will measure the binding efficiency and specificity to CD34+ cells with flow cytometry, and perform magnetic cell selection of spiked CD34+ cells in whole blood. If successful, the project will have significant ramifications because 1) it will significantly lower the cost of HSCT and, 2) it will advance the aptamer selection technology to rapidly generate high affinity aptamers against a wide range of cell surface markers, which have far-reaching application in clinical diagnosis and therapies. PUBLIC HEALTH RELEVANCE: Autologous hematopoietic stem cell transplantation (HSCT) has served as an important treatment option for many patients with hematologic malignancies and solid tumors including acute myeloid leukemia, myelodysplastic syndromes, chronic myeloid leukemia, systemic mastocytosis and breast cancer. Unfortunately, the antibody-based reagents used in the therapy to isolate CD34+ hematopoietic stem cells remain prohibitively expensive (>$10,000 per procedure). To address this issue, we aim to develop a novel system capable of rapidly generating aptamers, and utilize it to isolate high affinity aptamers that bind to the CD34 surface antigen. The success of this project has important ramifications as it will significantly reduce the cost for many cell-based transplantation therapies, and also establish a universal method to generate high-affinity aptamers for a wide range of diagnostic and therapeutic applications.